Novel Application of Reversed-Phase UPLC-oaTOF-MS for Lipid

Novel Application of Reversed-Phase UPLC-oaTOF-MS for Lipid Analysis in Complex Biological Mixtures: A New Tool for Lipidomics Paul D. Rainville,*,† Chris L. Stumpf,† John P. Shockcor,† Robert S. Plumb,† and Jeremy K. Nicholson‡ Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757, and Biological Chemistry, Division of Biomedical Sciences, Sir Alexander Fleming Building, Imperial College London SW7 2AZ, United Kingdom Received November 18, 2006

Ultra-Performance LC (UPLC) utilizing sub-2-µm porous stationary phase particles operating with high linear velocities at pressures >9000 psi was coupled with orthogonal acceleration time-of-flight (oaTOF) mass spectrometry and successfully employed for the rapid separation of lipids from complex matrices. The UPLC system produced information-rich chromatograms with typical measured peak widths of 3 s at peak base, generating peak capacities in excess of 200 in 10 min. Further UPLC coupled with MSE technology provided parent and fragment mass information of lipids in one chromatographic run, thus, providing an attractive alternative to current LC methods for targeted lipid analysis as well as lipidomic studies. Keywords: Lipidomics • Metabolic Profiling • Ultra Performance LC • Mass Spectrometry

Introduction Lipids are an important class of biomolecule that exist in great variety in higher organisms. The great diversity exhibited by these molecules is most probably due to the many biochemical functions in which these molecules are involved. Lipids are used as energy stores to fuel metabolism, as structural components in cell membranes, and are involved in many metabolic processes such as signal transduction, morphogenesis, secretion, and vesicle trafficking.1,2 Metabolic diseases such as Diabetes mellitus have been shown to have a direct relationship with a disorder in the lipids and fatty acids that make up phospholipids,3 a class of lipids that are defined by various specific polar head groups. This class of lipid has shown commercial use in biomembranes, skin care formulations, and in the making of liposomes, which are used in drug delivery as well as in cosmetics and detergents.3 The analysis and profiling of lipids has therefore become increasingly important in the fields of food analysis, commercial applications, and metabolic profiling. The ability to profile the lipids in biological fluids is also important, as it allows for the analysis of the effects of many candidate pharmaceuticals of metabolic pathways such as cholesterol synthesis. Lipidomics is a field of study that has rapidly expanded the study of lipids based on the advances made by electrospray ionization mass spectrometry4 that focuses on the study of lipid classes as they exist in their natural environment. The analysis of lipids has been performed by a diverse variety of approaches reflecting the diverse chemical subclasses. Gas chromatography (GC) and GC mass spectrometry (GC/MS) * Corresponding author. E-mail: [email protected]. † Waters Corporation. ‡ Imperial College London.

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approaches provide a rapid, sensitive, and relatively cheap method of analysis, but require prior time-consuming chemical derivatization which can leads to inter laboratory variations.5 1 H NMR provides a relatively fast method of profile analysis that can generate information on a range of lipids including lipoproteins, but the disadvantage of this approach is the difficulty in separating multiple overlapped lipidic species with the exception of lipoproteins. Lipids have been successfully analyzed by normal-phase high-performance liquid chromatography (HPLC)6-8 and normal-phase HPLC coupled with mass spectrometry;3,9-11 this method of analysis has high sensitivity and does not require any sample derivatization. However, the analysis times are typically long, 30-60 min, and peak resolution is typically poor, with peak width in the order of 20-30 s. Direct infusion of lipids into a mass spectrometer has also been shown as a method for characterization of cellular lipids,4 but this type of analysis of simultaneously introducing multiple analytes into a mass spectrometer can affect the detection of low-abundance species due to ion suppression. Reversed-phase HPLC has been the technique of choice for both pharmaceutical and bioanalytical LC/MS/MS analysis.12 This is due to the high efficiency of the separations, the compatibility of the mobile phase with biological and lipophilic samples, and the easy interfacing with a variety of detectors including UV, fluorescence, evaporative light scattering detection, radio-chemical detection, mass spectrometry, and NMR. In fact, a majority of liquid chromatographic separations used today are based upon reversed-phase LC. However, as previously stated, lipid analysis is often carried out using normalphase chromatography and becomes difficult if one is switching between the two different modes of chromatography on a single system. The process of changing from reversed-phase to normal-phase chromatography entails replacing not only the 10.1021/pr060611b CCC: $37.00

 2007 American Chemical Society

A New Tool for Lipidomics: Reversed-Phase UPLC-oaTOF-MS

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Figure 1. Reversed-phase UPLC separation of phospholipid standards shown with corresponding ESI negative MS specta. Shown are 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine, 3.9 min; 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], 8.20 min; and 1,2-dioleoyl-snglycero-3-phosphoethanolamine, 8.63 min. Table 1. Mass Accuracy of Phospholipids Standards Detected with Reversed-Phase UPLC/MS Method standard

1-oleoyl-2-hydroxysn-glycero-3phosphocholine 1,2-dioleoyl-snglycero-3[phospho-L-serine] 1,2-dioleoyl-snglycero-3phosphoethanolamine

retention theoretical measured ppm time mass (M - H) mass (M - H) error

3.90

566.3458

566.3463

-0.9

8.20

786.5285

786.5299

-1.8

8.63

742.5387

742.5399

-1.6

mobile phase, but also the wash and purge solvents. This task is time-consuming, as it is essential to remove all traces of water prior to the commencement of analysis, and can result in

several days of down time while the instrumentation become “dry”. Methods have been developed utilizing both aqueous13,14 and nonaqueous15,16 reversed-phase chromatography for the analysis of lipids, but to the authors’ knowledge, the analysis of lipids has not been shown using the high-resolution capabilities generated by ultra-performance liquid chromatography. Recent advances in porous particle manufacturing and chromatographic hardware has led to the generation of highresolution “Ultra Performance” liquid chromatography (UPLC). UPLC builds on the principles of HPLC but employs alkylbonded porous particles with a particle size less than 2 µm in diameter operated with increased mobile phase linear velocities. These small particles can generate significantly higher resolution separations than 5 or 3 µm stationary phases. This increased resolution results in superior separations, increased sensitivity, and faster analysis.12 Because of the increased Journal of Proteome Research • Vol. 6, No. 2, 2007 553

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Figure 2. Total ion current (TIC) chromatogram of UPLC/MS separation of analytes extracted from the supernatant of protein precipitated rat plasma; (A) electospray negative and (B) electrospray positive.

sensitivity and resolution that UPLC offers, it only seems logical to apply it to the field of lipid analysis.17 However, the smaller particles used in UPLC generate greater back pressure, making it necessary to have a chromatography system that can operate at pressures in excess of 10 000 psi. The need to handle elevated pressures in excess of 10 000 psi can be mitigated to some extent by the use of increased column temperature. This is possible because of the reduction in viscosity of the mobile phase solvents with increases in temperature. However, increases in column temperature can and often do result in a change in selectivity and peak elution order and as such is nonpredictable. Raising the column temperature also increases the optimal linear velocity required to run the column effectively, in turn, requiring the need to handle elevated pressures and negating the benefits of raising the column temperature. Here, we demonstrate the use of UPLC operated in reversed-phase mode at moderate temperatures for the analysis of lipids from rat plasma samples. We further show the utility of MSE, a technique whereby both precursor and fragment mass spectra are simultaneously acquired by alternating between high and low collision energy during a single chromatographic run.18 The advances presented here may be significant for lipidomic research, which has so far been technically limited to relatively low-throughput applications. 554

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Experimental Procedures Sample Preparation. Acetonitrile, chloroform, and methanol were all obtained from Fisher Scientific (NH). Ammonium acetate, acetic acid (spectroscopic grade), and leucine-enkephalin were purchased from Sigma/Aldrich (MO). Distilled water was purified “in-house” using a MilliQ system Millipore (MA). Phospholipid standards were obtained from Avanti Polar Lipids (AL) as dry powder and were reconstituted in chloroform and sonicated until dissolved. Rat plasma was obtained from Equitech-Bio, Inc. (TX). Lipids were extracted from rat plasma by precipitating rat plasma with 3:1 (acetonitrile/rat plasma) and centrifuging at 14 700g for 5 min. The supernatant was then removed and injected into the UPLC/MS system. Chromatography. Reversed-phase analysis was performed on a Waters ACQUITY Ultra Performance LC system using a ACQUITY UPLC BEH C8 1.7 µm, 2.1 × 100 mm, analytical column. The column was maintained at 60 °C and eluted using a linear gradient of 35-95% B over 9 min where A consisted of 50 mM ammonium acetate, pH 5.0, and B consisted of acetonitrile at a flow rate of 0.6 mL/min. Mass Spectrometry. Mass spectrometry was performed on a Waters Q-Tof Premier mass spectrometer operating in negative and positive ion electrospray mode. The capillary and cone voltages were set at 3.2 kV and 35 Vfor positive electrospray mode and 2.5 kV and 45 V for negative electrospray mode.


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Figure 3. (A) Fragmentation pattern of 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine. (B, top panel) High collision energy mass spectra of 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine during MSE experiment. (B, bottom panel) Low collision energy mass spectra of 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine during MSE experiment.

The desolvation temperature was set to 350 °C and the source temperature to 120 °C; the cone gas was set to a flow rate of 50 L/h, and the desolvation gas flow was maintained at 800 L/h. MSE analysis was performed on a Waters Q-Tof Premier mass spectrometer set at 5 eV for low collision energy and 25 eV for high collision energy. Data Analysis. Principal component analysis (PCA) was carried out using MarkerLynx software from Waters Corporation (MA). Orthogonal partial least-squares (OPLS) analysis was carried out using SIMCA-P software from Umetrics (Sweden).

Results and Discussion To illustrate the performance of the UPLC separation, a standard containing three phospholipids was injected into the UPLC system. The peak shape for all three phospholipids standards is excellent with little if any tailing, Figure 1. The average peak width for this separation is 3 s at peak base, giving a peak capacity of 200 for a 10 min separation. The mass spectrum and corresponding chemical structure of the phospholipid peaks is also displayed in Figure 1. The accurate mass measurement of these peaks is shown in Table 1. This data shows that it is possible to obtain a sufficient number of points across these narrow chromatographic peaks to obtain good ion statistics. To further illustrate the capability of the reversed-phase UPLC/MS system for the analysis of lipids, lipids were extracted

from rat plasma and analyzed with the same method parameters as the previous sample. Both positive and negative ion electrospray were utilized because not all lipid species may ionize effectively in one mode. The chromatograms produced are shown in Figure 2; again we can see that the UPLC system produced a high efficiency separation. We then coupled the UPLC with a Q-tof Premier mass spectrometer operating in electrospray positive MSE mode. Illustrated in Figure 3 are the chemical structure and the electrospray positive MSE spectra of 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine. Observed are fragmentation ions 184 and 104 m/z generated from the choline head group under high-energy conditions. An extracted ion chromatogram of the high collision energy scans shows that a vast majority of analytes present in supernatant from protein precipitated rat plasma contain ions 184 and/or 104 m/z, Figure 4. We then repeated this experiment using three different lots of rat plasma. PCA carried out using the top 10 most intense ions detected during the analysis showed separation of the different lots of rat plasma tested, indicating that a statistical difference exists between different lots of rat plasma based on the top 10 most intense ions, Figure 5. OPLS S plot further generated a list of ions (blue data points) responsible for the inter-lot difference, Figure 6. Ions calculated from the OPLS S plot as contributing the greatest to the inter-lot difference are shown in Table 2. UPLC/MSE analysis carried out on these ions listed in Table 2 generated high-energy spectra containing Journal of Proteome Research • Vol. 6, No. 2, 2007 555

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Figure 4. Electrospray positive extracted ion chromatogram (XIC) and TIC of UPLC/MS separation of choline-containing lipids extracted from rat plasma. (A) XIC of m/z 184 and 104 and (B) TIC.

Figure 5. PCA scores plot showing variability between three different lots of rat plasma. Six repeat injections from each lot are also shown.

major fragment ions of 184 and 104 m/z. An example of this is shown for precursor mass 496.3397, Figure 7. A database search of the monoisotopic precursor mass was done using the LIPID 556


MAPS Web site and produced two possible matches within an error of 1.3 ppm: 2-lyso glycerophospho-ethanolamine (2-lyso GPEtn) and 2-lyso glycerophosphocholine (2-lyso GPCho).

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Figure 6. OPLS S-plot, data point in blue indicate ions responsible for variation between lots 1 and 2 of protein precipitated rat plasma. Table 2. Top 10 Parent Mass Ions Contributing to Differences between Rat Plasma Lots 1 and 2 m/z

R.T.

weight

confidence

496.33 524.36 520.33 522.35 521.34 544.33 496.61 802.60 774.57 722.60

3.88 4.78 3.65 4.2 3.657 3.72 3.88 7.96 7.36 10.1

0.350881 0.292548 0.227726 0.141613 0.124123 0.114852 0.0983533 0.0842536 0.079154 0.0763954

0.992827 0.965764 0.99346 0.980016 0.987388 0.960234 0.996497 0.97337 0.997217 0.982884

These two database matches produce different diagnostic fragments associated with their headgroup. 2-Lyso GPCho is expected to produce ions of 184 and 104 m/z, while 2-lyso GPEtn is expected to produce ions of 141 m/z.19,20 The high collision spectrum in Figure 7 shows the diagnostic fragments identifying 2-lyso GPCho as the correct structural match from the database. This information gathered by the UPLC/MSE method coupled with the fact that lysophospholipids and phospholipids are known to exist in the supernatant proteinprecipitated plasma19 indicate that a majority of the analytes present in the supernatant of protein precipitated rat plasma are lipids that contain the choline group.

Conclusion Ultra-Performance Liquid Chromatography (UPLC) utilizing sub-2-µm porous stationary phase particles operating with high linear velocities at pressures in excess of 9000 psi was coupled with oaTOF mass spectrometry and successfully employed in the analysis of lipids from mammalian plasma. Chromatographic peak widths of 3 s at peak base were observed, thus, generating separations with peak capacities in excess of 200 in just 10 min. Accurate mass measurement of less than 2 ppm was observed

Figure 7. MSE spectra of 496.3397, the most statistically significant ion found by OPLS between protein precipitated rat plasma lots 1 and 2.

in the mass spectra showing the capability of obtaining good ion statistics for these narrow chromatographic peaks. UPLC coupled with Q-Tof MS operating in MSE mode gave both fragment and parent information of lipids in a single chromatographic run. MSE experimental results further were used to distinguish between two database matches for a single monoisotopic mass without further experimentation. Lastly, this method coupled with PCA and OPLS indicated that choline-containing lipids account for the variability observed between the supernatant of different lots of protein-precipitated rat plasma.

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Novel Application of Reversed-Phase UPLC-oaTOF-MS for Lipid

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